This project's goal is to use a well-characterized model system to define processes governing the formation and activity of CD4+CD25+Foxp3+ regulatory T (Treg) cells. The proposal will make use of existing lineages of transgenic mice expressing the influenza virus hemagglutinin (HA) under the control of a variety of promoters (HA Tg mice) and/or HA-specific T cell receptors with varying affinities and specificities for the HA (TCR Tg mice). By analyzing double transgenic mice co-expressing HA-specific TCRs and neo-self HA peptides (TCRxHA Tg mice), we have shown that highly specific interactions with HA-derived peptides can induce thymocytes to undergo selection to become Treg cells that suppress conventional CD4+ T (Tconv) cell responses in vitro, and can modify immune responses in vivo. We have shown that the formation of these Treg cells is highly sensitive to variations in the amount of the HA that is expressed in different HA Tg lineages, and that expression of HA selectively by antigen presenting cells in one of these lineages (designated HACII mice) induces the spontaneous development of inflammatory arthritis in TCRxHACII mice despite the presence of antigen-specific Treg cells. We will use this unique model system to examine how TCR specificity directs Treg cell formation and determines the capacity of Treg cells to modulate anti-self and anti-viral immune responses. In Aim 1 we will examine how TCR recognition of self-peptides shapes Treg repertoire formation. We will modulate the reactivity of the TCR for self-peptides and determine the effects on thymic Treg cell development in TCRxHA Tg mice, and will use adoptive transfer approaches to examine the development of Treg cells in the periphery of HA Tg mice under various conditions. In Aim 2 we will examine how Treg specificity for self-peptides shapes anti-viral immunity. We will analyze the ability of Treg cells from TCRxHA Tg mice to modulate antibody responses to influenza viruses with which they possess varying degrees of crossreactivity, and examine HA Tg mice that do not co-express TCR transgenes for their frequencies of virus- specific CD4+ Treg and Tconv cells. In Aim 3 we will determine how TCR specificity impacts the ability of Treg cells to prevent autoimmune arthritis. We will either deplete Treg cells, or add Treg cells with varying degrees of reactivity with the HA to pre-arthritic TCRxHACII mice, and determine the effects on cellular processes that accompany arthritis development. We will also analyze the expansion and differentiation of Treg cells with varying degrees of reactivity for the HA following transfer into HACII or TS1xHACII mice. These studies will provide fundamental insights into the mechanisms of immune repertoire formation and tolerance. They will enhance our understanding of the role immune regulation plays in anti-viral immunity and how its failure can contribute to autoimmunity, and will enhance the ability of Treg cells to b exploit in diagnostic therapeutic settings.